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Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay
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Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Published on: August 26, 2018

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Site-Specific mRNA Cleavage for Selective and Quantitative Profiling of Alternative Splicing with Label-Free Optical

Cesar S Huertas1,2, Sophie Bonnal3,4, Maria Soler1,5

  • 1Nanobiosensors and Bioanalytical Applications Group , Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, BIST and CIBER-BBN , 08193 Bellaterra, Barcelona , Spain.

Analytical Chemistry
|November 6, 2019
PubMed

Insights

This study introduces a novel method to analyze alternative mRNA splicing using RNase H and optical biosensors. This technique enables precise, real-time detection of specific mRNA fragments, improving cancer biomarker analysis.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Alternative splicing of mRNA precursors is crucial for proteome diversity and is often dysregulated in cancer, presenting potential biomarkers.
  • Label-free optical biosensors offer sensitive nucleic acid analysis but struggle with complex RNA structures hindering alternative splicing detection.
  • Existing methods for analyzing mRNA isoforms are often indirect or lack the sensitivity required for complex biological samples.

Purpose of the Study:

  • To develop a robust methodology for the direct and sensitive detection of alternative mRNA splicing events using label-free optical biosensing.
  • To overcome the challenge of long-range base-pairing interactions in mRNA that impede direct isoform analysis.
  • To enable real-time monitoring of alternative splicing for potential applications in cancer diagnostics.

Main Methods:

  • Generation of length-controlled RNA fragments from total RNA using RNase H enzyme activity guided by specific DNA oligos.
  • Hybridization of DNA oligos to target RNA sequences, followed by site-specific RNase H cleavage of flanking regions.
  • Real-time monitoring of generated mRNA fragments using a surface plasmon resonance (SPR) biosensor.

Main Results:

  • Demonstrated selective and specific detection of mRNA fragments in the picomolar to nanomolar concentration range.
  • Achieved 81% accuracy in alternative splicing detection compared to quantitative reverse transcription PCR (RT-qPCR).
  • Showed that site-specific RNA cleavage improved detection accuracy by 20% over random hydrolysis, reducing quantification errors.

Conclusions:

  • The developed methodology enables efficient, label-free quantification of alternative splicing events in complex samples.
  • This approach significantly enhances the accuracy and sensitivity of detecting mRNA isoforms, overcoming previous limitations.
  • The technique holds promise for advancing cancer biomarker discovery and real-time monitoring of gene regulation.

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